The generation of polarity in epithelial cells depends on the coupling of extracellular spatial cues to intracellular membrane traffic. Coupling reflects the ability of plasma membrane signals to induce an ordered reorganization of the cytoskeleton and membrane-associated scaffolds. In turn, these infrastructural elements must control the translocation, and fusion of transport vesicles on the secretory and endocytic pathways. The goal of this project is to elucidate how epithelial cells transduce and integrate spatial information to effect polarized transport of plasma membrane components. Four Specific Aims are planned: First, we plan to determine the spatial organization of post-Golgi and endosomal recycling pathways to the plasma membrane of polarized epithelial cells. Using advanced live cell imaging techniques, we will determine how cell-cell and cell-substrate contact controls the orientation of transport pathways to the plasma membrane in kidney (MDCK) cells and hepatocytes. Second, we will characterize the extent to which known and suspected components associated with polarized vesicle traffic act by controlling the translocation and targeting of post-Golgi transport vesicles. Various considerations suggest that specific microtubule motors, components of the exocyst complex, GTPases and their exchangers are involved in directing vesicles to their appropriate domains. Third, we will determine the role of junctional complex-associated components in transducing spatial cues important for cell polarity. Special attention will be devoted to a complex consisting of the PDZ proteins Par3 and Par6, Cdc42, and PKC-zeta, in part using a chemical genetic approach. We will determine the possible involvement of these and other components in orienting membrane traffic. Fourth, we will begin to evaluate the role of receptor tyrosine kinases in polarity. Recent evidence has indicated the importance of the proto-oncogenes ErbB2 and Met in cell migration, morphogenesis, and polarity. We will determine the roles played in specifying the spatial organization of the cytosol and the polarity of vesicle translocation in part using a chemical genetic approach.